Host cell death following virus infection can influence viral pathogenesis. The receptor-interacting protein kinase 3 (RIPK3) is a signaling node that controls cell death following virus infection. The goal of the proposed research is to illuminate a new signaling pathway that leads to RIPK3 activation and RIPK3-mediated necrotic cell death. Mammalian reovirus (MRV) elicits a RIPK3-dependent necrotic cell death program in some cell types. However, our preliminary data rule out the function of RIPK1, DAI (DNA-dependent activator of IFN- regulatory factors), and TIR-domain-containing adapter-inducing interferon-b (TRIF), the three possible factors that typically function upstream of RIPK3. Once RIPK3 is activated, it typically induces necrosis via the membrane-disrupting function of the mixed lineage kinase-like (MLKL) protein. Our studies indicate that though MLKL is activated by RIPK3 in MRV-infected cells, the absence of MLKL does not prevent cell death. Thus, signaling pathways that activate RIPK3 and those that execute cell death downstream of RIPK3 in MRV- infected cells are both unknown. Thus, additional studies on MRV-induced cell death have the potential to uncover new pathways upstream and downstream of RIPK3 that lead to cell death. In Specific Aim 1, we will use a whole genome pooled CRISPR screen to identify molecules required for MRV-induced necrosis. MLKL- deficient cells transduced with sgRNAs will be challenged with MRV. The death-resistant cell pool will be stained using anti-MRV antisera and sorted into those that support MRV infection and those that are refractory to MRV infection. Deep sequencing and quantitation of sgRNAs enriched in MRV-permissive, death-resistant cells in comparison to uninfected cells will identify those genes that are required for cell death. RIPK3- dependent cell death following MRV infection also requires synthesis of progeny dsRNA. Cell death can be inhibited by GuHCl treatment, which prevents synthesis of dsRNA. However, the mechanisms by which GuHCl influences viral RNA levels is not understood. In Specific Aim 2, we will select for GuHCl resistant mutants by passaging virus in the presence of increasing concentrations of GuHCl. Resistant viruses will be sequenced and rebuilt by reverse genetics to identify residues that confer GuHCl resistance. The capacity of mutant viruses to synthesize viral dsRNA and plus strand RNA will be quantified. RIPK3-dependent cell death can also be enhanced by knockdown of the viral 1 protein. How 1 exerts this effect is also not known. In Specific Aim 2, we will also determine the impact of 1 on viral RNA synthesis and on the exposure of dsRNA. Completion of the proposed studies will identify a new pathway for RIPK3-dependent cell death. This work will also reveal viral determinants that control the accumulation and detectability of viral dsRNA. In addition to fueling future mechanistic studies on RIPK3-dependent cell death, these studies could prove useful in our ability to modulate cell death to influence viral pathogenesis, and in the design of viral vectors for cancer therapy.